Back-Scattered Electron Imaging of Sections Through the Cochlea: A New Technique for Studying Cochlear Morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Ponceau-Fuchsin as a Routine Countebstain

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Back-scattered electron imaging of sections through the cochlea: a new technique for studying cochlear morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Light microscopic identification and photography of Golgi impregnated central nervous system neurons during sectioning for electron microscopy.

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin sections of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which part of the neuron and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.     

Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

Cytological Staining Procedures Applicable to Methacrylate-Embedded Tissues

Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast microscope, mitochondrial preparations become routinely available without the uncertainties of most of the mitochondrial staining methods. It appears, therefore, that plastic embedding should find a useful place among the methods for light microscopy as well as in the armamentarium of the electron microscopist.     

Light Microscopic Identification and Photography of Golgi Impregnated Central Nervous System Neurons During Sectioning for Electron Microscopy

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Dynamic alterations in luteinizing hormone-releasing hormone (LHRH) neuronal cell bodies and terminals of adult rats

Summary 1. The decapeptide lueteinizing hormone-releasing hormone (LHRH) is synthesized in neuronal cell bodies diffusely distributed across the basal forebrain and is secreted from neuronal terminals in the median eminence. Once secreted, LHRH enters the portal vessels and is then transported to the anterior pituitary, where it modulates the synthesis and secretion of gonadotropins, which are essential to gonadal function and reproduction.2. Because of the difficulties encountered in studying these diffusely distributed neurons, we have developed strategies which combine immunocytochemistry and computer-assisted techniques to examine individual LHRH neuronal cell bodies, as well as the entire population of LHRH neurons from the diagonal band of Broca to the mammillary bodies. In addition, we have examined LHRH neuronal terminals in the median eminence using computer-assisted imaging techniques to examine individual terminals by electron microscopy or across all rostral-caudal regions of the median eminence by light microscopy. In our most recent studies using confocal microscopy, we have examined the relationships of LHRH terminals to glial processes.3. These studies reveal a very dynamic system of LHRH neuronal cell bodies and terminals. The population of neurons in which LHRH can be detected varies as a function of time after gonadectomy, during the estrous cycle, and during the preovulatory surge of LH during the afternoon of proestrus. Dynamic changes are also observed in LHRH terminals in the median eminence as a function of time after gonadectomy and in specific rostral-caudal regions of the median eminence during the preovulatory surge of LH. Finally, confocal microscopy reveals that LHRH terminals are prevented from contacting the basal lamina of the brain by glial end-feet.4. We are currently examining the hypothesis that these relationships change as a function of endocrine milieu and, therefore, participate in the modulation of LHRH secretion. Ongoing studies focus on defining the sites of action and synergy of multiple sources of regulation of LHRH secretion and their relative importance to ensuring reproductive success.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

STUDIES ON NUCLEI USING CORRELATED CYTOCHEMICAL, LIGHT, AND ELECTRON MICROSCOPE TECHNIQUES

In this paper, a procedure for correlating electron microscope and light microscope cytochemical studies using immediately adjacent serial thin and thick sections has been described and discussed. This technique, combined with the Feulgen reaction for DNA, has been of particular value in framing and answering both general and specific questions about the nucleus. The results may be summarized as follows:— Apparent nuclear homogeneity in the electron microscope is not due to loss of DNA as evidenced by positive Feulgen reactions in such nuclei. Arrangement of Feulgen-positive material in chromosomes, heterochromatin, perinuclear and perinucleolar chromatin, etc., is similar to that customarily observed in the light microscope but this is not necessarily reflected in a cursory survey of the electron image. Careful comparison of light and electron images shows that fine differences in structure are associated with chromatin localization. Primary spermatocyte prophase chromosomes of crayfish have been positively identified by their Feulgen-positive nature. Core-like axial structures in such chromosomes have been observed (9) and are described further. A remarkable feature of spermiogenesis in the crayfish is an elaboration of the nuclear envelope of the spermatid accompanying the formation of what becomes a mass of convoluted membranes in the sperm. In the spermatid, perinuclear chromatin follows outpocketings of the nuclear envelope into the cytoplasm. In the early sperm, on the other hand, although the nuclear envelope is continuous with the system of convoluted membranes, the chromatin is distinct from it and is retained in the nucleus proper by some mechanism independent of the nuclear envelope. None of the above observations was apparent from the electron microscope images alone; they were possible only by virtue of the correlated cytochemical and electron microscope study of adjacent sections. The successful use of other cytochemical tests, such as the PAS reaction for certain carbohydrates, in such correlated studies is also described.     

小鼠海马锥体细胞树突棘形态的电镜三维重建

大多数神经元的复杂三维结构是很难直接观察的。激光扫描共聚焦显微镜技术结合染料标记技术可以重建神经元的三维形态,但精细结构的识别需要电子显微镜。利用透射电子显微镜技术,可以得到连续超薄组织切片的高分辨率图像,结合计算机支持的三维重建技术就可进一步获得神经细胞精细结构的三维信息。通过电镜三维重建技术对未成熟和成熟小鼠海马锥体细胞树突棘的形态进行了观察和分析,并对其关键步骤的操作技巧进行了重点说明。实验结果为进一步利用成像技术研究树突棘的结构、功能和可塑性提供了重要信息。     

Same serial section correlative light and energy-filtered transmission electron microscopy.

Correlative imaging of a specific cell with both the light microscope and the electron microscope has proved to be a difficult task, requiring enormous amounts of patience and technical skill. We describe a technique with a high rate of success, which can be used to identify a particular cell in the light microscope and then to embed and thin-section it for electron microscopy. The technique also includes a method to obtain many uninterrupted, thin serial sections for imaging by conventional or energy-filtered transmission electron microscopy, to obtain images for 3D analysis of detail at the suborganelle level.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

Electron microscopic study of immunoreactive LHRH perikarya with special reference to neuronal regulation

Summary In early postnatal rats, immunoreactive LHRH perikarya in the preoptic area were studied by light and electron microscopy. Synaptic junctions were found between the immunoreactive perikaryon or its process, and the immunonegative nerve fibers. The significance of these synapses is discussed in relation to possible mechanisms by which the activities of LHRH neurons are regulated.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.